This research proposal addresses the need within the orthopaedic community for a rapid, accurate and inexpensive method for determining whether or not orthopaedic specimens, such as synovial fluid, and other orthopaedic tissues, such as bone and bone marrow, are infected. Standard laboratory culturing and Gram stain testing to detect bacterial infections in articular joints have been particularly disappointing due to the relatively high frequency of false negative test results. The applicants are developing, testing and refining a molecular biological technique using the polymerase chain reaction (PCR) to provide rapid and accurate determination of the presence of microorganismal DNA in an orthopaedic specimen. The methodology involves a novel way of processing orthopaedic samples to achieve bacterial lysis and release of DNA, allowing the direct molecular detection of that DNA by PCR. Bacteria-specific PCR primers are being used that amplify the highly conserved, universal 16S ribosomal RNA (rRNA) gene unique to bacterial genomes. A preliminary study is being undertaken to test the efficacy of this diagnostic technology for detecting infection in synovial fluid. Initially, the proposed process is being tested with sterile synovial fluid inoculated with known concentrations of various orthopaedically relevant bacterial species. Secondly, the applicants propose to extend this methodology to test synovial fluid specimens from patients who have undergone total knee replacement (TKA) surgery, and who may have acquired bacterial infections in the affected joints. Also proposed are strategies designed to use PCR diagnostics for the determination of the species of the infectious agent. A statistical comparison will be performing comparing the applicants' PCR detection methodology with standard diagnostic modalities. Placement of a revision implant into a contaminated joint because of false negative preoperative microbiological data will result in an infected revision TKA, requiring additional surgery at great expense, in terms of medical costs and patient discomfort. It is suggested by the investigators that, with the application of highly sensitive PCR diagnostic technology, rapid and accurate identification of infected specimens could greatly assist in the establishment of surgical and/or antibiotic treatment regimens. Thus, it is speculated that this proposed research could provide a valuable methodology for the orthopaedic clinician, as well as the basic researcher, when either is attempting to determine unambiguously the presence and level of infectious agents in a clinical orthopaedic specimen.